白姑鱼胰蛋白酶的分离纯化及其性质分析

Purification and Characterization of Trypsin from White Croaker(Argyrosomus argentatus)

本研究从白姑鱼幽门垂组织中提取胰蛋白酶粗酶,然后经硫酸铵分级沉淀、DEAE-Sepharose Fast Flow阴离子交换层析及Sephacryl S-200凝胶过滤层析等分离纯化技术,得到一种阴离子型胰蛋白酶（命名为：Trypsin A）,并对该阴离子型胰蛋白酶进行鉴定及性质分析。结果表明：Trypsin A的分子质量约为28 k D,其最适反应温度为50℃,能在低于65℃条件下保持稳定;最适p H值为9.5,酸碱耐受范围为p H 9.5~11.0;Western blotting分析表明,白姑鱼阴离子型胰蛋白酶可以与抗鲫鱼胰蛋白酶抗体反应,符合胰蛋白酶活性区域的高度保守性;丝氨酸类蛋白酶抑制剂对白姑鱼胰蛋白酶具有抑制作用。

An anionic trypsin（named as trypsin A） from the pyloric caeca of white croaker（Argyrosomus argentatus） was purified by ammonium precipitation, DEAE-Sepharose Fast Flow anion exchange chromatography and Sephacryl S-200 gel filtration chromatography. The characteristics of trypsin A were investigated. The results showed that trypsin A migrated as a single protein band with a molecular weight of about 28 kD in SDS-PAGE. Trypsin A exhibited its optimal temperature at 50 ℃ and was stable below 65 ℃. Trypsin A revealed its optimal p H at 9.5 and was stable in the p H range from 9.5 to 11.0. The purified trypsin A was analyzed by western blotting using anti-common carp trypsin antibody, and the result showed that the active region of the trypsin was highly conservative. Serine proteinase inhibitors were effective against trypsin A.